1. Field of the Invention
The present invention is concerned with improved novel immunoadjuvants.
As is known in the field of immunology, multiple injections of a vaccine may be necessary to induce an immune response in a host sufficient to provide immunity because the viral or bacterial antigen contained in the vaccine is often cleared from the injection site too rapidly to permit an adequate immune response. Thus, immunological adjuvants have been added to vaccines in order to delay release of the viral or bacterial antigen and/or to stimulate the host's immunological response. However, many of the earlier immunological adjuvants which were employed had serious drawbacks, such as causing irritation at the site of injection. Accordingly, the art has long sought an immunological adjuvant which would be readily metabolized by the host without serious side effects, while at the same time delaying release of antigen and stimulating the immune response of the host.
One of the most active immunoadjuvants is Freund's Complete Adjuvant which is a water-in-oil emulsion consisting of 10% Arlacel A and 90% mineral oil containing whole killed mycobacterial cells. A vaccine is formulated with Freund's Complete Adjuvant by incorporating the antigen in the aqueous phase. Therapeutic applications of Freund's Complete Adjuvant, however, have been prevented due to accompanying toxic side effects, such as local granulomas, endotoxic shock, and adjuvant-induced polyarthritis. The minimal active structure of mycobacteria has been determined by Ellouz et al., Biochem. Biophys. Res. Commun., 59, 1317 (1974) and by Kotani et al., Biken J., 18, 105 (1975) to be a peptidoglycan fragment of the cell wall, more specifically, a muramyl dipeptide, namely, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP). The addition of synthetic MDP to an emulsion of Freund's incomplete adjuvant (90% mineral oil and 10% Arlacel A) containing an antigen increases the level of antibodies against the antigen (humoral response) and induces delayed hypersensitivity (cellular immunity).
The effects of various structural modifications of the dipeptidyl moiety of MDP or biological activity have been reported, although studies on the effects of modifications of the saccharide moiety have been limited.
2. Brief Description of the Prior Art
Ellouz et al., Biochem. Biophys. Res. Commun., 59, 1317-25 (1974) discloses that MDP is the minimal bacterial cell wall fragment having adjuvant activity.
Adam et al., Biochem. Bioyphys. Res. Commun. 72 339-346 (1976) dicloses various lactyl dipeptide modifications of MDP. All were less active than MDP except N-acetyl-muramyl-L-seryl-D-isoglutamine which was as active as MDP.
Kotani et al., Biken Journal, 18, 105-111 (1975) discloses that MDP has adjuvant activity in saline solution as well as in water-in-oil emulsion.
U.S. Pat. Nos. 4,082,735 and 4,082,736 disclose various MDP analogs obtained by acetylating the sugar hydroxyl groups, varying the amino acid constituents of the dipeptides, using alkyl or aryl amide substituents, and varying the acid ether linkage between the sugar and the dipeptide.
Belgian patent Nos. 852,348 and 852,349 disclose N-acetylmuramyl dipeptides wherein the dipeptide is either L-alanyl-D-isoglutamine or L-seryl-D-isoglutamine, and their esters and amides.
Japanese patent Nos. 2083506 and 2046020 disclose N-acetylmuramyl dipeptides acylated with fatty acids at the 6-position of the sugar moiety.
Japanese patent No. J5-2156-812 discloses MDP acetylated in the 6-position with mycolic acid.
Japanese patent No. J5-3077-011 discloses an analog of the preceding compound wherein glycine is substituted for L-alanine in the dipeptide moiety.
Belgian patent Nos. 834,753 and 834,754 disclose oil-free adjuvant compositions containing MDP and an MDP analog wherein D-glutamic acid is substituted for D-isoglutamine.
Japanese Patent No. 54-130,517 discloses MDP higher fatty acid esters in which the 6-O-position is substituted with C.sub.1-90 acyl, optionally unsaturated, branched, or substituted, and optionally containing additional hydroxyl, carboxyl, carbonyl, amino, methoxy, or cyclopropyl functional groups.
The improved novel immunoadjuvants of the present invention differ from the prior art immunoadjuvants based on MDP in that the C-1 hydroxy of the sugar is replaced by a deoxy function. The resulting compounds are, thus, no longer reducing sugars and exist only in the pyranoid form of the sugar residue.